Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift

Abstract

BackgroundUnderstanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated.MethodsA simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker.ResultsThe possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated.ConclusionThe method allows for the enrichment of red blood cells early after the invasion by P. falciparum parasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.

To bet or not to bet: deciphering cell to cell variation in protozoan infections

“…Another possible application is the use of microfluidics to enrich for particular phenotypes, such as the enrichment for early-ring stages of P. falciparum based on the non-inertial lift effect using the altered deformability of infected red blood cells (iRBC) as an intrinsic marker. This method of sorting cells would increase the sensitivity of further genomic or diagnostic analyses [51]. In addition to the above, it has recently been shown, using single cell RNA sequencing (RNA-seq), that individual cells display well-differentiated enrichment modules of coexpressed genes and differences in alternative splicing at the mRNA level [52].…”

Advances in microfluidics in combating infectious diseases

“…Furthermore, as RBC deformability is also a characteristic of other diseases such as sickle cell anemia, improvements in specificity is warranted. Geislinger and colleagues also enriched less deformable ring-stage iRBCs with an average enrichment factor of 4.3 and throughput of 12,000 cells/h based on a separation process driven by the non-inertial lift effect (Geislinger et al, 2014). Warkiani et al made use of the negative depletion approach to remove leukocytes from lysed blood before also using inertial focusing (Warkiani et al, 2015).…”

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

“…Similarly, treatment with the pore-forming toxin Streptolysin O has been described as a method to selectively remove erythrocytes infected with late-stage parasites [21]. Furthermore, a microfluidics device has been developed for the enrichment of ring-stage parasites [22]. Other more commonly used methods take advantage of the decreased density of infected erythrocytes containing more mature stages, such as density centrifugation using a Percoll or Nycodenz cushion, to separate erythrocytes infected with later stage parasites from erythrocytes infected with ring-stage parasites and uninfected erythrocytes [23-26].…”